Steroids have been used in treatment for a variety of inflammatory diseases as a powerful anti-inflammatory agent. Steroids have, however, various kinds of side-effects because of its hormonal actions and its application must be limited to relatively serious cases. Thus, the development of a novel drug having anti-inflammatory action comparative to that of steroids but much reduced side-effects has been most seriously awaited by the doctors treating inflammatory diseases.
As one of the mechanisms for steroids to manifest its anti-inflammatory action, it has been proposed that the synthesis of a protein which inhibits phospholipase A.sub.2 is induced in the inflammatory site to suppress the production of arachidonic acid caused by the enzyme, resultantly the formation of the arachidonic acid metabolites having proinflammatory activity is lowered [Flower et al. Nature 278, 456, (1979)]. The phospholipase A.sub.2 -inhibitory protein has been expected to have the activity satisfactory for the above-stated requirements and numerous researchers have tried the isolation and purification of the protein, and the evaluation on its inflammatory action.
Until now, however, no protein having powerful actions comparative to steroids has been found and the following two reasons have been cited as the causes:
(1) In the previous researches, the enzyme purified from pancreas has been utilized in the evaluation system for activity inhibition, but the protein inhibitors of phospholipase A.sub.2 purified from inflammatory sites has a different structure and different activities from phospholipase A.sub.2 originating from pancreas, and
(2) The inhibitor does not directly act on phospholipase A.sub.2, and an apparent inhibitory activity by its interaction with the substrate has been detected.
The inventors paid attention to these problems and purified the phospholipase A.sub.2 from the peritoneal exudate of rats with peritonitis caused by casein.
Additionally, the inventors have succeeded in purification of phospholipase A.sub.2 from human inflammation sites, namely the synovial fluid in humans with rheumatoid arthritis [WO89/05851 specification (Japanese Patent 325255/(1987), Hara et al, J. Biochem. 104, 326-328 1988), and clarified that phospholipase A.sub.2 purified from human inflammatory sites very well resembles phospholipase A.sub.2 purified from rat inflammation region in the activity and structure. In other words, both enzymes showed high specificity to phosphatidylethanolamin. On the contrary, the phospholipase A.sub.2 purified from pancreas does not exhibit such substrate specificity. The sequence of 34 amino acids on the N-terminal was compared and resultantly, the homology between the phospholipase A.sub.2 purified from rat inflammation sites and the phospholipase A.sub.2 purified from human inflammation sites was 67%, while it was only 45% between the phospholipase A.sub.2 purified from human inflammatory sites and the phospholipase A.sub.2 purified from pancreas.
In the meantime, the inventors have found a protein fraction which specifically inhibits the phospholipase A.sub.2 purified from rat peritoneal exudate, in the peritoneal wash of the rats to which dexamethasone was given. The inventors purified the fraction to obtain proteins of about 35 kDa and 40 kDa and determined the amino acid sequence of individual N-terminals [Japanese Patent laid-open No. S 63-246397 (1988) (Japanese Patent Application No. S 62-79693 (1987)].
Further, the inventors obtained, as a protein specifically inhibiting phospholipase A.sub.2 purified from synovial fluid of humans with rheumatoid arthritis, a protein of about 33 kDa as an enzymatic degradation products of human complement C3 and determined the amino acid sequence of the N-terminal (Japanese Patent Laid-open No. H 2-91100 (1990) (Japanese Patent Application No. S 63-242556 (1988)].
These inhibitory proteins did not exhibit the inhibitory activity against the pancreas phospholipase A.sub.2 at all. In other words, these inhibitory proteins are quite unique because they have activities which have overcome the two above-mentioned problems and can be expected to manifest much more powerful anti-inflammatory action than that of the previously isolated phospholipase A.sub.2 -inhibitory protein.
The complete amino acid sequence of the protein inhibiting the phospholipase A.sub.2 purified from inflammatory sites has not yet been elucidated. In addition, it is very extremely difficult to obtain a sufficient amount of the phospholipase A.sub.2 -inhibitory protein to evaluate the inflammatory action, when it is extracted and purified from the rat peritoneal wash or degradation products of human C3.
Meanwhile, according to the inventors, the N-terminal amino acid sequence of protein inhibitors of phospholipase A.sub.2 purified from inflammatory sites, which has been purified from rat peritoneal wash, has extremely high homology with a part of the amino acid sequence of human complement C3.alpha. chain and is presumably a protein similar to C3 dg or C3d, a degradation product of C3.
It has been known that human complement C3 is cleaved stepwise by protease in serum into C3dg or C3d in the final stage. In addition, it has been reported that a high level of C3dg is detected in the synovial fluid of humans with rheumatoid arthritis by an immunochemical method and the values are correlated to the severity of the disease (Nydegger et al., J. Clin. Invest., 59, 862-868, 1977).
Previously, Japanese Patent Laid-open No. H 2-91100 (1990) described above is cited as a document relating to the correlation between the complement C3 and the phospholipase A.sub.2 -inhibitory protein purified from inflammatory sites. The patent document, however, does not describe the complete amino acid sequence and the gene of the inhibitory protein at all.
Further, there is no description of the correlation between said inhibitory protein and C3, particularly C3dg, a degradation product of complement C3, and of the preparation process for said inhibitory protein by enzymatic treatment of serum.
The object of the invention is to give the complete amino acid sequence of protein inhibitors of phospholipase A.sub.2 purified from inflammatory sites clearly.
Another object of the invention is to clarify the DNA sequence coding the amino acid sequence of protein inhibitors of phospholipase A.sub.2 purified from inflammatory sites and to provide the preparation process for giving a large amount of the protein.